Although considerable primary sequence data have been developed for voltage-dependent sodium channels by this laboratory and others, and a number of detailed models have been proposed for channel tertiary structure, experimental data have been reported that relates directly to the three-dimensional conformation of the channel in the membrane. Given the difficulties encountered in obtaining x-ray crystallographic information for membrane proteins such as the sodium channel, it is likely that high-resolution structural data will still be some years in coming. In the meantime, studies relating structure to function require the best possible analysis of channel three-dimensional structure in order to proceed. Because of the indirect nature of most structural approaches short of crystallography, a number of different techniques will have to be brought to bear. Building on our recent biochemical studies of the skeletal muscle sodium channel, and our cloning of the primary sequence for the rat TTX-sensitive and TTX-resistant isoforms of the sodium channel and their human homologs, we propose here to continue a multidisciplinary approach to muscle sodium channel structure, and to begin an analysis of the relationship between this structure and its function. We will probe the organization of the sodium channel in the membrane with studies that focus on the membrane topography of specific segments of the primary sequence, on the organization of the large extramembrane regions in the channel structure, and on the structure of the compactly folded internal repeat domains. Specifically, we will complete our analysis of the location of sites of post-translational modification in the channel primary sequence. We will extend our detailed analysis of the location and kinetics of protease-sensitive regions in the channel structure to include a topographical analysis of these regions, and an analysis of their response to alterations in membrane potential or toxin binding. We will extend our use of monoclonal antibodies to probe the interaction and location of the extramembranous domains. We will use antibodies to interhelical loops or to foreign epitopes inserted into interhelical loops to begin an analysis of the folding pattern within the repeat domains. Finally, in collaboration with Dr. Richard Horn, we will begin an analysis of structure function relationships in the muscle channel that takes advantage of the characteristic differences in toxin binding, single channel conductance, and kinetics between the muscle TTX-sensitive and TTX-resistant channels. This analysis will take specific advantage of the availability of expressible full-length clones for the two rat skeletal muscle sodium channel isoforms. Chimeras will be constructed between the two clones and the partitioning of characteristic properties investigated. Initial leads will be pursued with site-directed mutagenesis. Special attention will be paid to the S5-S6 interhelical loop regions in light of their potential role in toxin binding, and their markedly different treatment in key models of channel structure.